Phosphorylation of peptide substrates for the catalytic subunit of cAMP-dependent protein kinase.

Abstract

The steady-state kinetic parameters for the phosphorylation of four peptides by the catalytic subunit of cAMP-dependent protein kinase were measured as a function of pH. For peptides containing the minimum consensus sequence, R-R-X-S-hyd (where hyd is a hydrophobic residue), the kcat/Kpeptide profile is bell-shaped with pK values of 6.4 and 9.4. Inhibition studies with the peptide LRRNAI indicate that the lower pK corresponds to an intrinsic pK on the enzyme, whereas the higher pK is perturbed upward by 1 pH unit. Viscosity studies verify that substrate stickiness accounts for the kinetic perturbation of the higher pK in kcat/Kpeptide. Substitution of the P-3 arginine with alanine (where serine is the P-site) yields a kcat/Kpeptide versus pH profile that is also bell-shaped, although both pK values are intrinsic acid dissociation constants of the enzyme. Replacement of the P-2 arginine with alanine removes the lower pK in the pH-rate profile without altering the higher pK. These results indicate that recognition of the P-2 arginine requires the ionization of an enzyme residue. This result implies that if the catalytic subunit mechanism involves general base catalysis, the ionization of this bse is not manifested in the pH-rate profiles.